© 1995 American Heart Association, Inc.
Articles |
Role of Lipoxygenase Metabolites in Ischemic Preconditioning
From the Laboratory of Molecular Biophysics (E.M., W.G., T.F.), National Institutes of Environmental Health Sciences, Research Triangle Park, NC, and the Department of Pathology (C.S.), Duke University Medical Center, Durham, NC.
Correspondence to Dr Elizabeth Murphy, Laboratory of Molecular Biophysics, National Institutes of Environmental Health Sciences, Research Triangle Park, NC 27709.
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Abstract Preconditioning with brief intermittent periods of ischemia before a sustained period of ischemia has been shown to reduce infarct size and improve recovery of function in rat hearts. The mediators of this protective response are unknown in rats. We tested the hypothesis that a lipoxygenase metabolite might be involved in preconditioning, since lipoxygenase metabolites such as 12-hydroperoxyeicosatetraenoic acid have been shown to increase K+ channel activity and to decrease Ca2+ channel activity, which could have a protective effect on ischemic injury. In support of this hypothesis, we report that the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA, 5 µmol/L) and eicosatetraynoic acid (7 µmol/L) added just before and during preconditioning blocked the protective effects of preconditioning on recovery of function during reflow after 30 minutes of global ischemia. In addition, these lipoxygenase inhibitors partially blocked the ability of preconditioning to attenuate the rise in cytosolic free calcium during sustained ischemia. We also investigated the effects of preconditioning on eicosanoid metabolism by using high-performance liquid chromatography and found that 12-hydroxyeicosatetraenoic acid (12-HETE), the stable product of the lipoxygenase pathway, was made during the preconditioning protocol and that 12-HETE accumulation was blocked by NDGA. Thus, there is a correlation between functional recovery after ischemia and stimulation of the lipoxygenase pathway of arachidonic acid metabolism before the sustained period of ischemia; inhibition of the lipoxygenase pathway eliminates the protective effect of preconditioning on recovery of function after ischemia.
Key Words: lipoxygenase • ischemic preconditioning • hydroxyeicosatetraenoic acid
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Introduction |
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Preconditioning with brief intermittent periods of ischemia has been shown to reduce ischemic injury.1 2 3 4 5 6 7 8 Preconditioning was originally described as a protective mechanism that reduced the area of necrosis after a subsequent longer period of ischemia.1 3 4 7 8 Subsequently, numerous studies have shown that "preconditioning" also can provide protection against other detrimental effects of ischemia, such as arrhythmias and postischemic contractile dysfunction,2 5 6 although it is possible that the mechanisms responsible for the protective effects on necrosis, recovery of function, and arrhythmias may be different. Liu et al9 have suggested that adenosine mediates the reduction in necrosis observed in preconditioned rabbit hearts. In support of this hypothesis, adenosine has been shown to mimic preconditioning,10 11 12 and many adenosine antagonists block the ability of preconditioning to reduce the area of necrosis, but interpretation of adenosine antagonist data is complicated by the observation that many adenosine antagonists also block glucose uptake.13 Thornton et al14 have also shown that pertussis toxin treatment inhibits preconditioning, implicating the involvement of Gi proteins in preconditioning. It has been proposed that adenosine acting through G proteins may increase K+ channel activity, thereby reducing action potential duration and sparing ATP.15 16 However, data supporting the involvement of K+ channels in preconditioning are mixed.17 18 In rabbit heart, glibenclamide blocks preconditioning when ketamine/xylazine is used for anesthesia but does not block preconditioning when pentobarbital is used.17
Even though preconditioning with brief intermittent periods of ischemia has been shown in rat heart to have a protective effect on infarct size,3 4 recovery of function,2 5 and arrhythmias6 19 that is of similar magnitude to the protective effects in other species, the mediators of the protective effect appear to be different. In contrast to numerous studies reporting that adenosine is involved in the protective effect of preconditioning in rabbit heart,9 12 the data overwhelmingly indicate that adenosine is not the mediator of preconditioning in rat heart.3 13 20 Similar to what is observed in other species, adenosine agonist administered to the rat heart before ischemia reduces infarct size3 and leads to improved recovery of function10 11 after ischemia. The ability of adenosine to improve functional recovery, however, is lost if the heart is perfused without adenosine for 5 minutes before the sustained period of ischemia,20 although a protective effect of an adenosine A1 receptor agonist on infarct size persists despite 10 minutes of perfusion without adenosine before the sustained period of ischemia.3 Thus, adenosine receptor stimulation has the potential to protect the rat heart from subsequent ischemic injury, at least transiently, but adenosine antagonists do not block the protection afforded by preconditioning in the rat heart.3 13 20
In the present study, we have investigated a different mechanism that may be involved in preconditioning in rat heart by using contractile dysfunction and ionic alterations as end points of preconditioning. We hypothesize that preconditioning triggers the activation of K+ channels and the inhibition of Ca2+ channels, which may be similar to the effect of adenosine on these channels even if adenosine is not the primary mediator of preconditioning in the rat heart. Recent studies in neuronal tissues show that a G protein–sensitive pathway, involving a lipoxygenase metabolite of arachidonic acid (AA) and possibly a phosphatase, activates a K+ channel and inhibits a Ca2+ channel.21 22 23 24 In addition, AA intermediates have been shown to modulate K+ channels in heart, with lipoxygenase and cyclo-oxygenase products having opposite effects.25 Furthermore, it is known that AA is produced in the ischemic heart via stimulation of phospholipase A2 (PLA2), making this second-messenger pathway plausible.26 27 The activation of PLA2 may be mediated by mechanisms involving G proteins and/or protein kinase C, both of which have been shown to activate PLA228 29 30 31 32 and both of which have been suggested to be involved in preconditioning.14 33 34 Therefore, we have investigated the hypothesis that a mechanism of preconditioning may involve activation of PLA2 during the brief periods of ischemia. We further postulate that the preconditioning protocol stimulates AA metabolism and alters the profile of eicosanoid metabolites, which affects ion transport and other cell functions and mediates the preconditioning response. In support of this hypothesis, we report that the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and eicosatetraynoic acid (ETYA) block the protective effects of preconditioning on postischemic contractile dysfunction in the isolated perfused rat heart. We also have evaluated the effect of preconditioning on eicosanoid metabolism by using reverse-phase high-performance liquid chromatography (HPLC) analysis and find that the stable end product of 12-lipoxygenase metabolism, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], is made during the preconditioning protocol and that the 12-hydroxyeicosatetraenoic acid (12-HETE) accumulation is blocked by NDGA.
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Isolated Perfused Rat Heart Preparation
Adult male Sprague-Dawley rats (Charles River Suppliers, Wilmington, Mass), weighing 200 to 300 g, were anesthetized with pentobarbital. The heart was excised, and the aorta was cannulated. Retrograde perfusion was started from a reservoir 90 cm above the aortic cannula. The nonrecirculating perfusate was a Krebs-Henseleit buffer containing (mmol/L) NaCl 120, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 1.25, NaHCO3 25, and glucose 11. The buffer was continuously aerated with humidified 95% O2/5% CO2 and was maintained at 37°C. In experiments in which cytosolic free calcium concentration ([Ca2+]i) was measured, loading with 300 mL of 5 µmol/L of the acetoxymethyl ester of 5F-BAPTA was started after 15 minutes of control perfusion. With typical flow rates of 10 to 15 mL/min, loading took 20 to 30 minutes. After loading with 5F-BAPTA, or after the 15-minute equilibration period in hearts not loaded with 5F-BAPTA, hearts were perfused with phosphate-free Krebs-Henseleit buffer (pH adjusted to 7.4 by adding HCl) for
30 minutes, during
which time the magnet was shimmed and several preischemic spectra were
acquired. Phosphate-free buffer was used to unambiguously assign the
inorganic phosphate peak to the intracellular space as required for
measuring pHi. The heart was placed in a standard 20-mm
nuclear magnetic resonance (NMR) tube with the apex of the heart 1
cm from the bottom of the tube. The perfusate was evacuated by a
variable-speed Masterflex peristaltic pump connected to polyethylene
tubing.
For assessment of contractile function, a latex balloon on the tip of a
polyethylene catheter was inserted through the left atrium into the
left ventricle. The catheter was connected to a Statham P23d pressure
transducer that was outside the magnet at the same height as the heart.
The balloon, inflated to give an end-diastolic pressure of
5 to 10 cm water, was maintained at a constant volume until just before
reflow following the sustained period of ischemia. At this point, the
balloon was deflated to reduce the "no-reflow" phenomena. After
2 to 3 minutes of reflow, the balloon was reinflated to 5 to 10 cm
of water to allow measurement of left ventricular developed pressure
(LVDP) during reflow. Initially, all hearts developed at least 100 cm
H2O peak systolic pressure at an end-diastolic
pressure of 
10 cm H2O. 5F-BAPTA loading has been shown
previously to buffer calcium transients and thereby decrease peak
systolic pressure to 25 cm H2O at an
end-diastolic pressure of 10 cm
H2O.35 Hearts were paced at 5 Hz by use of a
Grass stimulator with a silver wire inserted into the right ventricle;
an electronic filter was used to avoid radiofrequency interference by
the pacing wires.
Experimental Protocols
We were interested in measuring the effects of ischemia and
reperfusion on ions, ATP, and LVDP in hearts preconditioned in the
presence and absence of inhibitors of AA metabolism. We chose inhibitor
concentrations based on values found in the
literature,23 24 36 37 after confirming by HPLC that these
chosen concentrations inhibited the appropriate AA pathway. In this
series of experiments, there were six groups of hearts: one group was
subjected to 30 minutes of global ischemia, and five groups were
"preconditioned" with four cycles of 5-minute ischemia, each
separated by 5 minutes of reflow, and then subjected to 30 minutes of
sustained global ischemia. One group was preconditioned with no added
drug, a second group was preconditioned in the presence of 5 µmol/L
NDGA, a third group was preconditioned in the presence of 4 µmol/L
clotrimazole, a fourth group was preconditioned in the presence of 3
µmol/L indomethacin, and a fifth group was preconditioned in the
presence of 7 µmol/L ETYA. After the 30-minute ischemic period, all
groups were reperfused for 20 minutes with phosphate-free
Krebs-Henseleit buffer without any added drug. Measurements of LVDP and
the 31P NMR data were obtained from hearts not loaded with
5F-BAPTA. In the experiments without 5F-BAPTA, 7 hearts were in the
untreated group, 12 hearts were in the preconditioned (no added drug)
group, 6 hearts were in the group preconditioned in the presence of
NDGA, 5 hearts were in the preconditioned clotrimazole-treated group, 5
hearts were in the preconditioned indomethacin-treated group, and 5
hearts were in the preconditioned ETYA-treated group. The inhibitors,
added to the perfusate 10 minutes before preconditioning, were
present during preconditioning and ischemia. On reflow, hearts were
reperfused without the inhibitors. To measure
[Ca2+]i, hearts were loaded
with 5F-BAPTA, and the same protocols were used. We studied 7 hearts in
the untreated group, 15 hearts in the group preconditioned without
drug, 5 hearts in the group preconditioned in the presence of NDGA, 5
hearts in the preconditioned clotrimazole-treated group, 6 hearts in
the preconditioned indomethacin-treated group, and 7 hearts in the
preconditioned ETYA-treated group.
To evaluate whether lipoxygenase inhibitors had detrimental effects during control perfusion or on recovery of contractile function after ischemia in nonpreconditioned hearts, an additional three groups were studied. Hearts were perfused for 30 minutes (with or without drug), followed by 15 minutes of ischemia and 20 minutes of reperfusion (without drug). In addition, to evaluate whether clotrimazole was beneficial in the absence of preconditioning, we also included a group treated with clotrimazole before ischemia. Five hearts were treated without drug, 6 hearts were treated with 5 µmol/L NDGA, 5 hearts were treated with 7 µmol/L ETYA, and 5 hearts were treated with 4 µmol/L clotrimazole. The duration of ischemia used for these studies was chosen to provide a moderate degree of stunning in the untreated hearts, so that beneficial or detrimental effects of drug treatment could be detected.
Measurement of Arachidonic Acid Metabolites
In another series of experiments, we measured AA metabolites in
hearts extracted with chloroform/methanol. In this series of
experiments, there were four groups of hearts, and hearts were not
loaded with 5F-BAPTA. The first group (n=6) was perfused for 30 minutes
with 10 µmol/L AA and then preconditioned with 5 minutes of ischemia,
5 minutes of reflow, and 5 minutes of ischemia (IRI protocol). The
second group (n=3) was treated with 5 µmol/L NDGA for 15 minutes,
perfused with 5 µmol/L NDGA and 10 µmol/L AA for 30 minutes, and
then preconditioned (IRI). The third group (n=3) was treated with 4
µmol/L clotrimazole for 15 minutes, perfused with 4 µmol/L
clotrimazole and 10 µmol/L AA for 30 minutes, and then preconditioned
(IRI). The fourth group (n=6) was perfused with 10 µmol/L AA for 30
minutes, followed by 30 minutes of ischemia. All hearts were frozen in
liquid nitrogen and subsequently extracted in chloroform/methanol by
the method of Bligh and Dyer.38 Organic extracts were
evaporated to dryness under argon and reconstituted in 50% methanol
(pH 3.5) for analysis by reverse-phase HPLC.
[3H]prostaglandin B2 was added to each sample
to serve as an internal standard.
Reverse-phase HPLC analyses were conducted with a C18 Ultrasphere column (5 µm, 4.6x250 mm, Altex Scientific) equipped with a Waters model U6K injector and a Waters model 6000A pump. Separation of eicosanoids was achieved by elution with a stepwise methanol gradient (55% to 100%, pH 5.05) at a flow rate of 1.1 mL/min as described previously.39 This HPLC system provides an effective separation of all classes of arachidonate metabolites (ie, prostaglandins, leukotrienes, diHETEs, HETEs, and free fatty acids) and is especially applicable for recovery and resolution of eicosanoids generated in biological systems. The effluent was monitored with a Waters model 900 photodiode array UV detector. UV-absorbing fractions were collected and subjected to further analysis.
For steric analysis of the hydroxy-AA metabolites, samples and
standards were converted to methyl esters by dissolving the material in
50 µL of methanol and then adding 200 µL of ethereal diazomethane.
After reaction for 2 minutes at room temperature, the samples were
evaporated to dryness under argon and reconstituted for further HPLC
analysis. For use as a chromatographic standard, milligram
quantities of racemic 12-HETE were prepared via controlled
auto-oxidation of AA in the presence of 
-tocopherol.40
The resulting hydroperoxides were reduced with triphenylphosphine to
the corresponding alcohols and were separated by semipreparative
straight-phase HPLC by using a Waters µPorasil column and a solvent
system of hexane/2-propanol/acetic acid (100:1.6:0.1 [vol/vol/vol])
with a flow rate of 4.0 mL/min. For chiral-phase HPLC, we used a
Pirkle-type dinitrobenzoyl phenylglycine column (5 µm, 4.6x250 mm,
Regis Chemical Co) with a mobile phase consisting of hexane/2-propanol
(100:1 [vol/vol]) at a flow rate of 2.0 mL/min.
NMR Procedures
NMR studies were performed on a Nicolet NT 360 wide-bore NMR
spectrometer with the variable temperature probe at 37°C. For the
19F studies, we used a 20-mm 19F probe tuned to
339.7 MHz (Doty Scientific). We used a 20-mm broad-band probe (Nicolet)
tuned to 146.1 MHz for the 31P NMR studies. The sample was
shimmed on the water signal from the heart, and we routinely obtained a
(nonspinning) line width at half height of 0.25 ppm. For the
19F studies, we used a 40° pulse angle, a ±5-kHz
spectral width, a 205-ms recycle time, and 4000 data points. A 70°
pulse angle, a 1-second delay, a ±5-kHz spectral width, and 4000 data
points were used for the 31P NMR studies.
As shown previously,41 42 Ca2+ binding to 5F-BAPTA exhibits slow exchange kinetics at 8.5 T, and [Ca2+]i is related to the observed resonance intensities by [Ca2+]i=Kdx[Ca2+ 5F-BAPTA]/[5F-BAPTA] where the Kd value for 5F-BAPTA is 700 nmol/L at 37°C42 and [Ca2+ 5F-BAPTA] and [5F-BAPTA] are proportional to the areas under their respective resonance peaks. Since this measurement requires the comparison of resonance intensities, it is necessary to work under conditions of nonsaturation of the resonances or, alternatively, under conditions of equal saturation. Fortunately, 5F-BAPTA and Ca2+ 5F-BAPTA have nearly identical spin lattice relaxation times41 ; therefore, the intensity ratio of free 5F-BAPTA to calcium-complexed 5F-BAPTA is essentially independent of the rate of pulsing. Studies by Marban et al43 show that in perfused hearts 5F-BAPTA is not significantly compartmentalized into mitochondria or endothelial cells. Resonance intensities were determined by digitizing the spectra by use of commercial software. pHi was measured from the shift between intracellular inorganic phosphate and creatine phosphate as described previously by Jacobus et al.44
Materials
NDGA, ETYA, clotrimazole, indomethacin, and -tocopherol
were obtained from Sigma. AA was obtained from NuCheck Prep.
12(S)-HETE standard was acquired from Cayman Chemical Co.
Triphenylphosphine was purchased from Aldrich Chemical Co. Solvents
used included HPLC-grade methanol, chloroform, 2-propanol, water, and
acetic acid from Baker, with hexane and ether from Mallinckrodt.
Ethanol was from Pharmco. 5F-BAPTA was obtained from Molecular
Probes.
Statistics
Values are expressed as mean±SEM. Data were analyzed by using
commercial software (SYSTAT). Time-matched data were
evaluated by ANOVA for repeated measurements. When F values indicated
that significant differences were present, we used methods that
adjust for multiple comparisons (Tukey's honestly significant
difference) to compare individual times for significant differences. A
value of P<.05 was considered significant.
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Preconditioning has been shown previously to improve recovery of function on reflow after 20 to 30 minutes of sustained ischemia in perfused rat and rabbit hearts.2 5 45 To investigate the role of metabolites of AA in preconditioning, we first evaluated whether inhibitors of AA metabolism affected the ability of preconditioning to improve postischemic contractile dysfunction. As shown in Fig 1
, hearts subjected to 30 minutes of
ischemia recovered only 20% of their preischemic LVDP. Hearts
preconditioned by four cycles of 5 minutes of ischemia and 5 minutes of
reflow and then subjected to 30 minutes of ischemia recovered 60%
of their preischemic LVDP. Hearts preconditioned in the presence of
lipoxygenase inhibitors such as NDGA and ETYA did not show improved
LVDP after 30 minutes of ischemia. Thus, these lipoxygenase inhibitors
were able to block the protective effects of preconditioning on
recovery of LVDP. Preconditioning in the presence of the
cyclo-oxygenase inhibitor indomethacin or the cytochrome P-450
inhibitor clotrimazole slightly, but insignificantly, enhanced the
recovery of LVDP after 30 minutes of ischemia.
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P<.05 vs no
PC, PC+NDGA, and PC+ETYA.
To establish that the ability of lipoxygenase inhibitors to eliminate
the protective effect of preconditioning on postischemic contractile
function is not due to a nonspecific effect of the drug on
contractility, coronary flow, or metabolism, we evaluated the effects
of ETYA and NDGA on LVDP, pHi, and ATP during 30
minutes of aerobic perfusion, 15 minutes of ischemia, and 20 minutes of
reflow in nonpreconditioned hearts. Thirty minutes of perfusion with
either drug did not significantly change LVDP or the coronary flow
rate. As shown in Fig 2, recovery of LVDP after 15
minutes of ischemia was not significantly reduced by 30 minutes of
perfusion with either ETYA or NDGA at the same concentrations as used
in Fig 1
. In addition, as shown in Fig 3, neither ETYA
nor NDGA significantly altered pHi during 30 minutes of
aerobic perfusion, and neither affected the fall in pHi
during 15 minutes of ischemia. During reflow, there was good recovery
of pHi in all groups, although the ETYA-treated group had a
slightly lower pHi after 20 minutes of reperfusion than the
other groups. Similarly, ATP levels were not significantly altered by
the presence of NDGA or ETYA during this protocol (data not shown).
Thus, there is no evidence that NDGA or ETYA is detrimental to the
heart under aerobic conditions or during a single episode of ischemia
and reperfusion. We also investigated whether clotrimazole can improve
postischemic recovery of contractile function in nonpreconditioned
hearts. As illustrated in Fig 2, in clotrimazole-treated hearts,
postischemic recovery of LVDP was 70% of initial LVDP compared with
55% in untreated hearts; the difference is not statistically
significant. In addition, clotrimazole treatment had no significant
effect on pHi (Fig 2) or ATP (data not shown) during
ischemia.
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The observation in Fig 1 that lipoxygenase inhibitors blocked the
protective effects of preconditioning is consistent with the hypothesis
that a lipoxygenase metabolite(s) might mediate preconditioning. If
this hypothesis is correct, one would expect that preconditioning
should lead to an increase in lipoxygenase metabolism. To investigate
whether lipoxygenase metabolites are produced during preconditioning,
we performed reverse-phase HPLC analysis on chloroform/methanol
extracts of hearts perfused with 10 µmol/L AA and subjected to the
IRI protocol. As shown in Fig 4A, the HPLC chromatogram
of the organic extract from these hearts (IRI) showed a prominent
UV-absorbing (235-nm) peak with a retention time of 70 minutes, labeled
2. This peak cochromatographs with the authentic 12-HETE standard and
has a characteristic conjugated diene chromophore UV spectrum
consistent with 12-HETE (see inset, Fig 4A). As shown in Fig 4A, these
extracts also contained a small peak with a retention time of 40
minutes, labeled 1, which eluted in the region of the dihydroxy-AA
metabolites, and a UV spectrum suggesting that it is a conjugated diene
rather than a conjugated triene. As shown in Fig 4B, pretreatment with
the cytochrome P-450 inhibitor clotrimazole blocked the accumulation of
the dihydroxy-diene (peak 1 in Fig 4A) but enhanced the accumulation of
the putative 12-HETE (peak 2 in Fig 4A and 4B). This suggests that the
dihydroxy-diene (peak 1 in Fig 4A) is a cytochrome P-450–mediated
hydroxylation product of the putative 12-HETE. Indeed, peak 1 has a
similar elution time in this reverse-phase HPLC system to 12,20-diHETE
standard (a known cytochrome P-450–derived metabolite of
12-HETE).46 47 48 We recovered insufficient amounts of
material for further structural characterization of this compound (peak
1). Fig 4B shows an additional peak (labeled 3), which has a retention
time and UV spectrum consistent with clotrimazole. Furthermore, as
shown in Fig 4C, pretreatment with the lipoxygenase inhibitor NDGA
blocked the biosynthesis of both compounds, consistent with the
hypothesis that 12-HETE is lipoxygenase metabolite. In addition,
ischemic hearts that were not preconditioned (30 minutes of AA followed
by 30 minutes of ischemia) showed little or no 12-HETE (data not
shown). Taken together, these data support the contention that the
preconditioning protocol stimulates the production of 12-HETE. The
12-HETE appears to be a lipoxygenase metabolite, and the
dihydroxy-diene compound appears to be a cytochrome P-450 metabolite of
12-HETE.
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To further document that compound 2 in Fig 4A and 4B is 12-HETE and
that it is a product of the lipoxygenase pathway, we performed the
experiment shown in Fig 5 by using chiral-phase HPLC.
12(S)-HETE is the primary isomer formed by the lipoxygenase
pathway, whereas the cytochrome P-450 pathway would generate
12(R)-HETE. The fraction corresponding to 12-HETE was
collected from reverse-phase HPLC and further purified by
straight-phase HPLC. To establish the configuration of the chiral
center of this hydroxy-AA compound, the purified material was converted
to the methyl ester derivative and analyzed by chiral-phase HPLC on a
dinitrobenzoyl phenylglycine column with UV monitoring of the diene
chromophore at 235 nm. As demonstrated in Fig 5, the chiral-phase HPLC
method clearly resolved racemic 12-HETE into two peaks (first panel),
whereas authentic 12(S)-HETE standard eluted as a single
symmetrical chromatographic peak (second panel). Moreover, coinjection
of 12(R,S)-HETE and 12(S)-HETE standards
demonstrated that the S enantiomer is the earlier eluting
compound (third panel). The rat heart–derived material eluted as a
slightly asymmetrical peak, with the majority of the product clearly in
the S configuration (75% [S] and 25%
[R]) (fourth panel). This product profile is indicative of
a lipoxygenase-type reaction.
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Having demonstrated that 12-HETE is made in the early preconditioning period, that it is primarily 12(S)-HETE, and that lipoxygenase inhibitors block the appearance of 12-HETE and also eliminate the protective effect of preconditioning on recovery of function after 30 minutes of ischemia, we next investigated whether lipoxygenase inhibitors would alter the ability of preconditioning to attenuate the ionic derangements that occur during ischemia. We have shown previously that preconditioned hearts show a smaller decline in pHi and less rise in [Ca2+]i during the 30-minute sustained period of ischemia. If a lipoxygenase metabolite such as 12-hydroperoxyeicosatetraenoic acid (12-HpETE), the precursor of 12-HETE, is the mediator of preconditioning and if reduction of these ionic alterations is involved in the protection provided by preconditioning, then it might be expected that the addition of lipoxygenase inhibitors would block the ability of preconditioning to reduce the ionic alterations. Thus, during sustained ischemia, hearts preconditioned in the presence of lipoxygenase inhibitors would show a decline in pHi and a rise in [Ca2+]i similar to that observed in nonpreconditioned hearts. Alternatively, if the ionic alterations are not an integral part of the protective effect of preconditioning, then the inhibitors might have no effect on the ionic alterations. For instance, we2 and others49 50 suggested previously that the attenuation of the fall in pHi might be due to glycogen depletion and therefore to reduced anaerobic glycolytic flux, in which case the lipoxygenase inhibitors would have little or no effect on the fall in pHi. We also postulated that the rise in [Ca2+]i was coupled indirectly to the fall in pHi. If this is correct, then the effects on [Ca2+]i should parallel the effects on pHi.
Measurement of the effects of lipoxygenase inhibition, cyclooxygenase
inhibition, and cytochrome P-450 inhibition on the fall in
pHi during 30 minutes of sustained ischemia in
preconditioned hearts is shown in Fig 6. The decline in
pHi during sustained ischemia in hearts preconditioned in
the presence of NDGA or ETYA was similar to that in hearts
preconditioned with no drug; neither lipoxygenase inhibitor was able to
block the effect of preconditioning on pHi alterations
during ischemia. We did observe, however, that hearts preconditioned in
the presence of clotrimazole had an even smaller decline in
pHi during ischemia than hearts preconditioned without
drug. At 5 and 10 minutes of ischemia, clotrimazole-treated hearts had
a pHi that was significantly higher than that in hearts
preconditioned with no drug. However, after 10 minutes of sustained
ischemia, the difference between clotrimazole-treated preconditioned
hearts and hearts preconditioned without drug was not statistically
significant. Hearts preconditioned in the presence of the
cyclo-oxygenase inhibitor indomethacin had a decline in pHi
during ischemia that was similar to that in preconditioned hearts (no
drug). The recovery of pHi in all groups during reperfusion
was nearly complete, and the recovery of creatine phosphate was not
significantly different, suggesting that differences in postischemic
contractile function are not due to lack of adequate reperfusion in the
drug-treated groups.
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Fig 7 shows the effect of NDGA, ETYA, indomethacin, and
clotrimazole on ATP content during the preconditioning period, the
sustained 30-minute period of ischemia, and reflow. The changes in ATP
before, during, and after the sustained period of ischemia were not
significantly altered by the inhibitors.
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Fig 8 shows the effect of the various inhibitors of AA
metabolism on the rise in [Ca2+]i
during the 30-minute sustained period of ischemia. Since the
acquisition is not gated to the cardiac cycle, the measurements of
[Ca2+]i are a time average of
[Ca2+]i in diastole and systole.
During sustained ischemia in nonpreconditioned hearts,
[Ca2+]i rises above 2 µmol/L by 20
minutes of ischemia. In hearts that are preconditioned,
[Ca2+]i rises much more slowly; at 20
minutes of ischemia, [Ca2+]i is <1
µmol/L. In the present study,
[Ca2+]i in preconditioned hearts after
20 minutes of sustained ischemia is 884±118 nmol/L compared with the
control time-averaged [Ca2+]i of
647±31 nmol/L. Hearts preconditioned in the presence of lipoxygenase
inhibitors such as NDGA and ETYA have a
[Ca2+]i during sustained ischemia that
is intermediate between preconditioned and nonpreconditioned hearts. As
shown in Fig 8, at 20 minutes of sustained ischemia, hearts
preconditioned in the presence of ETYA have a
[Ca2+]i of 1468±232 nmol/L, lower
than the value of 2520±438 nmol/L in nonpreconditioned hearts but
higher than the [Ca2+]i in hearts
preconditioned with no drug (884±118 nmol/L). Hearts preconditioned in
the presence of NDGA also showed a
[Ca2+]i during sustained ischemia that
was intermediate between preconditioned and nonpreconditioned hearts;
at 20 minutes of sustained ischemia in hearts preconditioned in the
presence of NDGA, [Ca2+]i was
1617±385 nmol/L (data not shown). Hearts preconditioned in the
presence of clotrimazole had [Ca2+]i
of 774±186 nmol/L after 20 minutes of sustained ischemia. It is
unclear, however, to what extent the attenuated rise in
[Ca2+]i in the presence of
clotrimazole is due to inhibition of cytochrome P-450 metabolism versus
the reported ability of clotrimazole to inhibit calcium
influx.51 Hearts preconditioned with indomethacin had
[Ca2+]i values similar to hearts
preconditioned with no drug. After 20 minutes of sustained ischemia,
hearts preconditioned in the presence of indomethacin had
[Ca2+]i of 951±249 nmol/L (data not
shown). Similarly, at 25 minutes of ischemia,
[Ca2+]i in nonpreconditioned hearts
(2693±148 nmol/L) was significantly higher than in preconditioned
hearts (1205±87 nmol/L), significantly higher than in
clotrimazole-treated preconditioned hearts (718±138 nmol/L),
significantly higher than in indomethacin-treated preconditioned
hearts, but not significantly different than in ETYA-treated
preconditioned hearts (1750±347 nmol/L) or NDGA-treated preconditioned
hearts (2044±402 nmol/L). Thus, the inhibitors of the lipoxygenase
pathway of AA metabolism have a much greater effect on the rise in
[Ca2+]i during the sustained period of
ischemia than on the fall in pHi.
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Role of Lipoxygenase Metabolites in Preconditioning
The lipoxygenase inhibitors NDGA and ETYA both blocked the preconditioning-induced improvement in LVDP during reperfusion after 30 minutes of ischemia. Because of concerns about the specificity of inhibitors,36 37 we measured the correlation between lipoxygenase metabolites and preconditioning. We also used two separate lipoxygenase inhibitors, and we were careful to use these inhibitors in concentrations that have been reported to be specific for the inhibition of lipoxygenases.36 Data in the present study show that 12(S)-HETE, a stable alcohol generated via peroxidase reduction of 12-HpETE, is made early in the preconditioning protocol. Only trace amounts of 12(S)-HETE were occasionally observed with 30 minutes of ischemia, without reperfusion, in nonpreconditioned hearts. We postulate that 12(S)-HETE is generated by the lipoxygenase pathway since its formation is blocked by NDGA and enhanced by clotrimazole. The demonstration in Fig 5
that the
hydroxy chiral center is predominantly in the S
configuration is further evidence that 12-HETE is a product of the
lipoxygenase pathway. We further postulate that 12(S)-HETE
is metabolized to an unidentified compound (peak 1 in Fig 4A), which
has a retention time consistent with a dihydroxy-AA metabolite and a UV
spectrum indicative of a conjugated diene chromophore. This compound
appears to be formed by the cytochrome P-450 pathway, since
clotrimazole blocks its biosynthesis and enhances the accumulation of
12(S)-HETE. Further support for the hypothesis that the
dihydroxy-diene is a cytochrome P-450–derived hydroxylation metabolite
of 12(S)-HETE is shown in Fig 4C; when the formation of
12(S)-HETE is blocked by NDGA, no dihydroxy-diene compound
is made. Thus 12(S)-HETE is formed during early
preconditioning, and its biosynthesis is blocked by a lipoxygenase
inhibitor (NDGA) and enhanced by a cytochrome P-450 inhibitor
(clotrimazole). The appearance of 12(S)-HETE during the
preconditioning protocol correlates with improved recovery of function
after 30 minutes of ischemia. 12(S)-HETE is observed in
preconditioned hearts in the absence or presence of clotrimazole; in
both these conditions, there is excellent recovery of function after 30
minutes of sustained ischemia (60% to 75%). In contrast, little
12(S)-HETE is observed in nonpreconditioned hearts and in
hearts preconditioned in the presence of NDGA, both of which are
associated with poor recovery of function (<20%). The lipoxygenase inhibitors do not significantly alter pH, ATP, or LVDP during 30 minutes of aerobic perfusion, do not affect the fall in pHi or the rate of ATP depletion during 15 minutes of ischemia, and do not impair the recovery of LVDP significantly during reperfusion after 15 minutes of sustained ischemia. The postischemic contractile dysfunction, however, tended to be worse in lipoxygenase-treated hearts, consistent with the hypothesis that lipoxygenase metabolites exert a beneficial effect when present during ischemia or the early reflow period. However, the benefit of lipoxygenase metabolites is likely to be greater when there are intermittent brief periods of ischemia before a sustained period of ischemia; the brief periods of ischemia can stimulate the metabolic pathways, but the presence of oxygen is required for HpETE production, which is available during the periods of reperfusion. In hearts that are not preconditioned, there would be limited oxygen during ischemia to generate lipoxygenase metabolites. Thus, blocking the lipoxygenase pathway would have less impact in hearts that are not preconditioned. Although clotrimazole-treated hearts tended to have slightly improved postischemic contractile function compared with untreated hearts, the difference was not significant and may be related to inhibition of calcium influx rather than an effect on AA metabolism.
We had shown previously that preconditioning attenuates the rise in
[Ca2+]i and the decline in
pHi that occur during the sustained 30 minutes of ischemia.
Since lipoxygenase inhibitors block the protective effects of
preconditioning on postischemic functional recovery, it might be
expected that they would also block the attenuation of the ionic
alterations observed with preconditioning. We observed that without
preconditioning, pHi falls to 6.0 by 20 minutes of
ischemia; in preconditioned hearts, pHi falls only to 6.4
by 20 minutes of ischemia. If a lipoxygenase metabolite is responsible
for reducing the decline in pHi in preconditioned hearts,
then one would expect that hearts preconditioned in the presence of
NDGA would show a decline in pHi similar to
nonpreconditioned hearts; instead, we observed that pHi
fell to 6.3 after 20 minutes of ischemia, a value not significantly
different from that in the hearts preconditioned with no drug. This
suggests that a lipoxygenase metabolite is not responsible for the
effect of preconditioning on the fall in pHi during the
sustained period of ischemia.
A major difference between preconditioned and nonpreconditioned hearts
is that the fall in pHi stops abruptly after 10 minutes
in preconditioned hearts but continues for an additional 5 to 10
minutes in nonpreconditioned hearts. This could reflect glycogen
depletion in the preconditioned hearts, which would limit the amount of
anaerobic glycolysis that can occur and could account for the
difference in pHi at the end of 30 minutes of sustained
ischemia between preconditioned and nonpreconditioned hearts.
Consistent with this concept, a recent study showed that the difference
in the fall in pHi can be eliminated if the time between
the preconditioning protocol and the sustained period of ischemia is
increased to allow resynthesis of glycogen.50 This
suggests that intracellular acidification during sustained ischemia in
preconditioned hearts is limited by the amount of glycogen available at
the start of the sustained period of ischemia, which would be similar
in all preconditioned hearts, regardless of inhibitor treatment. Taken
together, the data suggest that preconditioning has multiple
consequences: preconditioning reduces glycogen, which limits anaerobic
glycolytic flux and reduces H+ accumulation, and in
addition, preconditioning increases the production of
12(S)-HETE, which may reduce Ca2+ channel
activity and/or increase K+ channel activity. This is
consistent with the effect of lipoxygenase inhibition on the rise in
[Ca2+]i during the sustained period of
ischemia. After 20 minutes of ischemia in hearts preconditioned in the
presence of ETYA, [Ca2+]i rose to 1.5
µmol/L, a value higher than in hearts preconditioned without drug
(0.9 µmol/L) but less than the 2.5 µmol/L measured in
nonpreconditioned hearts. This suggests that the rise in
[Ca2+]i during ischemia is partly
dependent on the fall in pHi and partly dependent on a
mechanism that is independent of pHi possibly mediated by a
lipoxygenase metabolite of AA. The data further suggest that recovery
of LVDP correlates better with the rise in
[Ca2+]i than the decline in
pHi.
In lipoxygenase inhibitor–treated preconditioned hearts, [Ca2+]i rises during the sustained period of ischemia to a value intermediate between untreated preconditioned hearts and nonpreconditioned hearts; however, postischemic contractile dysfunction in lipoxygenase-treated hearts is very similar to nonpreconditioned hearts. This could be explained if poor postischemic contractile function is the result of exceeding a threshold level of [Ca2+]i. Alternatively, it is possible that the lipoxygenase inhibition has some detrimental effect that is not mediated by [Ca2+]i: either blocking a beneficial effect mediated by a lipoxygenase metabolite, a nonspecific effect of the inhibitors, or a cumulative effect of the brief periods of ischemia and the sustained period of ischemia. If our hypothesis is correct, HpETE produced during the first period of ischemia and reflow would be protective during the second period of ischemia, and the additional HpETE produced during each subsequent cycle of ischemia and reperfusion could further enhance the protective effect. Blocking HpETE production could allow some injury to occur during the second, third, and fourth ischemic period of the preconditioning protocol as well as during the sustained period of ischemia.
Role of Eicosanoids as Effector Molecules
There are considerable data indicating that lipoxygenase
metabolites are involved in the activation of ion channels in
neurons21 22 23 24 and myocytes.25 52 53 Piomelli
et al23 have shown that lipoxygenase metabolites of AA
mediate the response of Aplysia neurons to FMRFamide. These
investigators showed that the lipoxygenase metabolite 12-HpETE mimics
FMRFamide; addition of 12-HpETE (1.5 µmol/L) to the cells stimulated
a slow membrane hyperpolarization and a decrease in excitability,
responses similar to those obtained with FMRFamide.23
5-HpETE, 5-HETE, and 12-HETE had little effect on resting
potential.23 Buttner et al22 showed that the
increase in the probability of S-K+ channel opening with
FMRFamide is mimicked by application of 12-HpETE to cell-free membrane
patches that lack ATP and GTP; this demonstrates that 12-HpETE can act
directly to modulate these channels independent of G proteins and
protein kinases. Buttner et al tested the effects of 12-HpETE in
inside-out and outside-in patches and found that 12-HpETE applied to
the outside of the membrane produced a larger, more rapid increase in
channel activity and was effective at a 10-fold lower concentration
(725 nmol/L). These data suggest that 12-HpETE might be released from a
cell and act on a neighboring cell. However, 12-HpETE does modulate
these channels when added to the inside,22 although
metabolism of 12-HpETE may be required.54 Other
lipoxygenase metabolites are also suggested to modulate ion channel
activity.55 Leukotrienes are reported to mediate the
somatostatin augmentation of the M current in hippocampal
cells.24 Similarly, leukotrienes have been reported to be
involved in the regulation of K+ channels in atrial
cells.52 In addition, AA and lipoxygenase metabolites
have been reported to alter Ca2+ transport across
mitochondria and sarcoplasmic reticulum and to alter
[Ca2+]i.56 57 58 59 Cytochrome
P-450 metabolites of AA are also reported to alter cell calcium
regulation.60 61 In addition, there are reports that
Ca2+ channels, including K+-activated
Ca2+ channels, are inhibited by cytochrome P-450
inhibitors, such as clotrimazole, at the same concentrations that
inhibit cytochrome P-450–dependent metabolism.51 It is
suggested that a cytochrome P-450 metabolite is involved in the
regulation of these channels.51 We observed no effects of
clotrimazole on LVDP under aerobic conditions, but it is possible that
inhibition of these channels may help explain the beneficial effects of
clotrimazole on ion changes during ischemia and contractile dysfunction
during reflow.
Parratt and coworkers62 63 have reported that the cyclo-oxygenase inhibitor meclofenamate blocks the preconditioning-induced reduction in arrhythmias in dogs. Other investigators have found that cyclo-oxygenase inhibition does not eliminate the protection against infarction afforded by preconditioning in the rabbit64 and does not block the protective effects of preconditioning on infarct size and arrhythmias in the rat.65 Our data are in agreement with the later studies,64 65 which have reported that cyclo-oxygenase inhibitors do not block the protective effects of preconditioning. The cyclo-oxygenase inhibitor used in the present study, indomethacin, may also function as a free radical scavenger. Neither action of the drug altered the beneficial effects of preconditioning on ionic changes during the sustained period of ischemia or on postischemic recovery of contractile function.
What Do We Know About the Mechanism of Preconditioning?
In general, it appears that preconditioning involves G proteins;
that agonists such as adenosine or acetylcholine, which activate G
proteins, mimic precondition-ing10 11 12 14 ; and that
pertussis toxin blocks preconditioning.14 In addition,
activation of protein kinase C is implicated since there are reports
that phorbol esters can mimic preconditioning33 34 and
that inhibitors of PKC can block preconditioning.33 There
are also reports that stretch can activate
preconditioning66 and that free radical scavengers can
block preconditioning.67 We postulate that preconditioning
activates a pertussis toxin–sensitive G protein, which activates
PLA2. Protein kinase C, which has been shown to translocate
to the plasma membrane during early ischemia,34 68 may
also be important in "priming"
PLA2.30 32 Activated PLA2 leads to
the release of AA, which is metabolized by the lipoxygenase pathway to
form 12-HpETE, which can activate K+ channels and
inactivate Ca2+ channels.21 22 23 24 It is
likely that some messenger might alter the activity of key enzymes in
the metabolism of AA (see Reference 3232 ), thus altering the profile of
eicosanoids and bringing about many changes that together we call
preconditioning. Eicosanoid metabolites have been shown to induce
numerous changes in cells.21 22 23 24 25 52 53 54 55 56 57 58 59 60 61 This hypothesis is
consistent with the observation that phorbol ester can mimic and
staurosporin can block preconditioning, since phorbol esters have been
shown to activate PLA2.29 30 32 This model is
also consistent with the pertussis toxin sensitivity of
preconditioning. Oxidation of lipid by free radicals has also been
shown to activate PLA2,32 providing a
possible explanation for the observation that free radical scavengers
may, in some cases, reduce preconditioning. Related to this,
lipoxygenase inhibitors such as NDGA are known to be excellent
antioxidants. Furthermore, the report that stretch can mimic
preconditioning is consistent with the observation that stretch
activates PLA2.69 70 71 These data might also
provide an explanation for the contrast between rabbit heart, which
appears to rely on adenosine as the mediator of preconditioning, and
rat heart, which demonstrates protective effects of preconditioning
even in the presence of adenosine receptor antagonists. Perhaps
adenosine is the sole activator of PLA2 and the
12-lipoxygenase pathway in rabbit and other species during brief
myocardial ischemia, but some other signal activates PLA2
and the 12-lipoxygenase pathway during preconditioning in rat
heart.
Although this hypothesis proposes that PLA2 stimulation may be involved in generating the mediator of preconditioning, previous studies generally have attributed detrimental consequences to PLA2 activity during myocardial ischemia.27 Phospholipid hydrolysis could contribute to ischemic injury by generating lysophospholipids, which may be arrhythmogenic and may have a detergent-like effect on membranes, or by depleting the membrane of phospholipids. However, these actions may require longer durations of ischemia than are used to induce the preconditioning phenomenon. Furthermore, cellular phospholipases are diverse enzymes that reside in different cellular compartments, are activated by different mechanisms, and have different pH optima. It appears that it is the cytosolic PLA2 that is responsible for the metabolism of AA to effector eicosanoids.30 Phosphorylation and calcium have been shown to cause translocation of cytosolic PLA2 to the plasma membrane; this translocation coincides with the activation of PLA2.30 32 This may be the critical event in the generation of the eicosanoid metabolites responsible for the protective effects of preconditioning. Furthermore, Miller et al72 have shown recently that inhibition of the cytosolic PLA2 does not appear to block cell injury.
This hypothesis also appears to be at odds with studies showing that lipoxygenase inhibitors provide protection from ischemia and reperfusion.73 74 75 It should be noted, however, that these cited studies used an in vivo model of ischemia and reperfusion with a duration of ischemia that is sufficient to produce infarction, resulting in neutrophil infiltration during reperfusion and generation of 12-HETE among other mediators. In addition, the protocol in many of these studies involved examining the capacity of ischemic/reperfused tissue to generate 12-HETE when incubated with A23187. Thus, these investigators are using a different model and are investigating the role of leukocytes and platelets in reperfused infarcts to generate lipoxygenase metabolites. Lipoxygenase metabolites may contribute to ischemia/reperfusion injury under these circumstances (in the presence of neutrophils) and may contribute to lethal injury when isolated cardiac myocytes are incubated under hypoxic conditions for 45 minutes and then reoxygenated, although free radical generation may play a larger role than the lipoxygenase metabolites.76 However, this does not preclude the possibility that a lipoxygenase metabolite may function as a mediator of preconditioning. It is possible that preconditioning is triggered by a compound that in larger doses or over a longer time period could be detrimental. One would not consider ischemia to be beneficial, yet brief periods of ischemia and reflow obviously trigger mechanisms that are protective. We postulate that it is a precursor of 12-HETE that is likely to mediate preconditioning. Furthermore, since lipoxygenases require O2, it is unlikely that significant lipoxygenase metabolism occurs during ischemia alone but rather that it occurs primarily during reoxygenation.76 This would explain the necessity of reoxygenation during the preconditioning protocol; reoxygenation would allow generation of lipoxygenase metabolites, which could then trigger preconditioning.
In summary, the data in the present study show that lipoxygenase inhibitors block the protective effects of preconditioning and the generation of 12(S)-HETE. These data are consistent with a role for lipoxygenase metabolites in preconditioning.
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Acknowledgments |
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C. Steenbergen was supported in part by National Institutes of Health grant HL-39752.
Received June 9, 1994; accepted November 7, 1994.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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